Activated dual specificity lymphocytes and their methods of use

ABSTRACT

The present invention relates to preventive, therapeutic, and diagnostic compositions and methods employing lymphocytes having T-cell receptors and chimeric receptors. In particular, the invention relates to pre-selected dual-specificity lymphocytes having endogenous T-cell receptors and chimeric T-cell receptors that recognize a strong antigen and tumor associated antigens where the pre-selected population of adoptively transferred lymphocytes is activated by in vivo immunization, thereby increasing the effectiveness of adoptive immunotherapy.

FIELD OF THE INVENTION

The field of the present invention relates generally to compositions andmethods for the treatment or prevention of diseases in mammals. Morespecifically, this invention relates to pre-selected dual-specificitylymphocytes having endogenous T-cell receptors and/or chimeric T-cellreceptors that recognize a strong antigen and tumor associated antigensand to preventative, diagnostic and therapeutic applications whichemploy these lymphocytes.

BACKGROUND OF THE INVENTION

Classic modalities for the treatment of diseases such as human cancers,autoimmune diseases, viral, bacterial, parasitic and fungal diseasesinclude surgery, radiation chemotherapy, antibiotics or combinationtherapies. However, these therapies are not effective against a majorityof these diseases. Alternate therapies for preventing or treating humandiseases are greatly needed. In the past decade immunotherapy and genetherapy utilizing T-lymphocytes have emerged as new and promisingmethods for treating human disease, in particular human cancers.

The T cell receptor for antigen (TCR) is responsible for the recognitionof antigen associated with the major histocompatibility complex (MHC).The TCR expressed on the surface of T cells is associated with aninvariant structure, CD3. CD3 is assumed to be responsible forintracellular signaling following occupancy of the TCR by ligand.

The T cell receptor for antigen-CD3 complex (TCR/CD3) recognizesantigenic peptides that are presented to it by the proteins of the majorhistocompatibility complex (MHC). Complexes of MHC and peptide areexpressed on the surface of antigen presenting cells and other T celltargets. Stimulation of the TCR/CD3 complex results in activation of theT cell and a consequent antigen-specific immune response. The TCR/CD3complex plays a central role in the effector function and regulation ofthe immune system.

Two forms of T cell receptor for antigen are expressed on the surface ofT cells. These contain either α/β heterodimers or γ/δ heterodimers. Tcells are capable of rearranging the genes that encode the α, β, γ and δchains of the T cell receptor. T cell receptor gene rearrangements areanalogous to those that produce functional immunoglobulins in B cellsand the presence of multiple variable and joining regions in the genomeallows the generation of T cell receptors with a diverse range ofbinding specificities. Each α/β or γ/δ heterodimer is expressed on thesurface of the T cell in association with four invariant peptides. Theseare the γ, δ and ε subunits of the CD3 complex and the zeta chain. TheCD3 γ, δ and ε polypeptides are encoded by three members of theimmunoglobulin supergene family and are found in a cluster on humanchromosome 11 or murine chromosome 9. The zeta chain gene is foundseparately from other TCR and CD3 genes on chromosome 1 in both themouse and human. Murine T cells are able to generate areceptor-associated η chain through alternative splicing of the zetamRNA transcript. The CD3 chains and the zeta subunit do not showvariability, and are not involved directly in antigen recognition.

All the components of the T cell receptor are membrane proteins andconsist of a leader sequence, externally-disposed N-terminalextracellular domains, a single membrane-spanning domain, andcytoplasmic tails. The α, β, γ and δ antigen-binding polypeptides areglycoproteins. The zeta chain has a relatively short ectodomain of onlynine amino acids and a long cytoplasmic tail of approximately 110 aminoacids. Most T cell receptor α/β heterodimers are covalently linkedthrough disulphide bonds, but many γδ receptors associate with oneanother non-covalently. The zeta chain quantitatively forms eitherdisulphide-linked ζ-η heterodimers or zeta-zeta homodimers.

Another example of a type of receptor on cells of the immune system isthe Fc receptor. The interaction of antibody-antigen complexes withcells of the immune system results in a wide array of responses, rangingfrom effector functions such as antibody-dependent cytotoxicity, mastcell degranulation, and phagocytosis to immunomodulatory signals such asregulating lymphocyte proliferation, phagocytosis and target cell lysis.All these interactions are initiated through the binding of the Fcdomain of antibodies or immune complexes to specialized cell surfacereceptors on hematopoietic cells. It is now well established that thediversity of cellular responses triggered by antibodies and immunecomplexes results from the structural heterogeneity of Fc receptors(FcRs).

FcRs are defined by their specificity for immunoglobulin isotypes. Fcreceptors for IgG are referred to as FcγR, for IgE as FcεR, for IgA asFcαR, etc. Structurally distinct receptors are distinguished by a Romannumeral, based on historical precedent. Three groups of FcγRs,designated FcγRI, FcγRII, and FcγRIII are now recognized. Two groups ofFcεR have been defined; these are referred to as FcεRI and FcεRII.Structurally related although distinct genes within a group are denotedby A, B, C. Finally, the protein subunit is given a Greek letter, suchas FcγRIIIAα, FcγRIIIAγ.

Considerable progress has recently been made in defining theheterogeneity for IgG and IgE Fc receptors (FcγR, FcεR) through theirmolecular cloning. These studies make it apparent that Fc receptorsshare structurally related ligand binding domains, but differ in theirtransmembrane and intracellular domains which presumably mediateintracellular signaling. Thus, specific FcγRs on different cells mediatedifferent cellular responses upon interaction with an immune complex.The structural analysis of the FcγRs and FcεRI has also revealed atleast one common subunit among some of these receptors. This commonsubunit is the γ subunit, which is similar to the ζ or η chain of theTCR/CD3, and is involved in the signal transduction of the FcγRIII andFcεRI.

The low affinity receptor for IgG (FcγRIIIA), is composed of the ligandbinding CD16α (FcγRIIIAα) polypeptide associated with the γ chain(FcγRIIIAγ). The CD16 polypeptide appears as membrane anchored form inpolymorphonuclear cells and as transmembrane form (CD16™) in NK. TheFcγRIIIA serves as a triggering molecule for NK cells.

Another type of immune cell receptor is the IL-2 receptor. This receptoris composed of three chains, the α chain (p55), the β chain (p75) andthe γ chain. When stimulated by IL-2, lymphocytes undergo proliferationand activation.

Antigen-specific effector lymphocytes, such as tumor specific T cells(Tc), are very rare, individual-specific, limited in their recognitionspectrum and difficult to obtain against most malignancies. Antibodies,on the other hand, are readily obtainable, more easily derived, havewider spectrum and are not individual-specific. The major problem ofapplying specific antibodies for cancer immunotherapy lies in theinability of sufficient amounts of monoclonal antibodies (mAb) to reachlarge areas within solid tumors. In practice, many clinical attempts torecruit the humoral or cellular arms of the immune system for passiveanti-tumor immunotherapy have not fulfilled expectations. While it hasbeen possible to obtain anti-tumor antibodies, their therapeutic use hasbeen limited so far to blood-borne tumors [Lowder, J. N. et al. CancerSurv. 4:359-375 (1985); Waldmann, T. A. Science 252:1657-1662 (1991)]primarily because solid tumors are inaccessible to sufficient amounts ofantibodies [Jain, R. K. J. Natl. Cancer Inst. 81:64-66 (1989)]. The useof effector lymphocytes in adoptive immunotherapy, although effective inselected solid tumors, suffers on the other hand, from a lack ofspecificity (such as in the case of lymphokine-activated killer cells(LAK cells) [Mule, J. J. et al. Science 225:1487-1489 (1984)] which aremainly NK cells) or from the difficulty in recruiting tumor-infiltratinglymphocytes (TILs) and expanding such specific T cells for mostmalignancies [Rosenberg, S. A. et al. Science 233:1318-1321 (1986)].Yet, the observations that TILs can be obtained in melanoma and renalcell carcinoma tumors, that they can be effective in selected patientsand that foreign genes can function in these cells [Rosenberg, S. A. J.Clin. Oncol. 10:180-199 (1992)] demonstrate the therapeutic potentialembodied in these cells.

A strategy which has been developed (European Published PatentApplication No. 0340793) allows one to combine the advantage of theantibody's specificity with the homing, tissue penetration, cytokineproduction and target-cell destruction of T lymphocytes and to extend,by ex vivo genetic manipulations, the spectrum of anti-tumor specificityof T cells. Chimeric T cell receptor (cTCR) genes composed of thevariable region domain (Fv) of an antibody molecule and the constantregion domain of the antigen-binding TCR chains, i.e., the α/β or γ/δchains have been expressed in T cells and found to be functionallyactive. Adoptive immunotherapies using tumor infiltrating lymphocytesand IL-2 have been developed for some cancers. These therapies haveresulted in significant long-term responses in some patients withmelanoma.

In an effort to broaden the applicability of adoptive immunotherapy tocommon cancers, such as, for example, ovarian, breast and colon cancertreatments that redirect the immune reactivity of lymphocytes toantigens recognized by monoclonal antibodies have been developed. To dothis, retroviral vectors that encode chimeric receptor genes consistingof the variable regions of a monoclonal antibody joined to thetransmembraneous and cytoplasmic domains of a T-cell receptor (TCR)signaling chain have been utilized. Using this approach, the safety ofthe administration of these chimeric receptor-transduced lymphocytes hasbeen demonstrated. However, a need for improving the effectiveness ofthe chimeric receptor-transduced lymphocytes exists.

Thus, one object of the present invention is to produce an activatedchimeric receptor-transduced lymphocyte capable of binding to andobliterating cancer cells.

SUMMARY OF THE INVENTION

One particular object of the present invention is to increase theeffectiveness of adoptive immunotherapy by increasing the persistenceand/or activity of adoptively transferred T cells by activating apre-selected population of adoptively transferred lymphocytes with invivo immunization.

Another objective of the invention is to create dual specific T cells bygenetic modification so that each individual T cell is reactive withboth a strong antigen, such as, for example an alloantigen or otherforeign agent, and the tumor. The strong antigen is used both to expandand activate the cells in vitro and in vivo.

The present invention relates to a composition comprising a populationof T cells transduced with a chimeric receptor gene and pre-selected forreactivity with a strong antigen. Another embodiment of the presentinvention comprises a lymphocyte having a TCR directed to a specificstrong antigen and a chimeric T-cell receptor directed to a tumorantigen, wherein the lymphocyte has been activated in vivo by the strongantigen. Such cells exhibit strong anti-tumor response and provide arecipient of such cells with a protection from tumor challenge, i.e.prophylactic response, and an anti-tumor treatment.

The method of treating a patient with therapeutically-activateddual-specificity lymphocytes comprises the steps of expanding apatient's lymphocytes with one or more specific strong antigens ex vivo,transducing the lymphocytes with a chimeric receptor gene, introducingthe transduced lymphocytes into the patient and immunizing the patientwith the strong antigents) in vivo. A preferred embodiment of thepresent method utilizes an alloantigen as the strong antigen. Anotherpreferred embodiment utilizes a virus or other foreign antigen as thestrong antigen.

The present invention also relates to a method of treating a patientwith dual specificity lymphocytes having reactivity to one or morepre-selected strong antigens comprising the steps of administering aneffective amount of such lymphocytes to a patient and immunizing thepatient with the strong antigen.

The present invention relates to activated dual specific lymphocytescontaining chimeric genes suitable to endow lymphocyte cells withantibody-type specificity and T-cell receptors specific for one or morepre-selected strong antigens. Various types of lymphocytes are suitable,for example, natural killer cells, helper T cells, suppressor T cells,cytotoxic T cells, lymphokine activated cells, subtypes thereof and anyother cell type which can express chimeric receptor chain.

The present invention further relates to pharmaceutical, prophylacticand curative compositions containing an effective quantity of suchcells.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a model of the dual-specific T cell created by geneticmodification such that each individual T cell has specificity for boththe strong antigen/immunogen and tumor. The chimeric receptor enablestumor recognition and comprises single-chain antibody variable regions(scFv) connected to TCR signaling chains, such as the γ chain of the Fcreceptor.

FIG. 2 shows the results of treating 8 advanced ovarian cancer patientswith MOv-PBL infusion. Specifically, treatment was measured by thepercentage of circulating MOv-γ transduced lymphocyte cells followingMOv-PBL infusion.

FIGS. 3A and 3B show the in vivo effects of allogeneic immunization ofadoptively transferred T cells in mice spleen, lung, and blood uponimmunization with allo-splenocytes (A) and alto-DC (dendritic cells; B)as a measure of Thy 1.1 cells in tissue.

FIG. 4 shows the phenotypic results of dual specific mouse T cells,where these T cells express the chimeric MOv-y.

FIG. 5 shows that mice are protected against tumor challenge wheninfused with dual specific T cells followed by immunization withallogeneic splenocytes.

FIG. 6 shows a time course graph of the adoptive transfer of dualspecific T cells followed by immunization treatment of establishedsubcutaneous tumor in immunodeficient mice. The results show that dualspecific T cells inhibit tumor growth and that the effect is augmentedby immunization.

FIG. 7 demonstrates that dual specific MOv-γ transduced human T cellscapable of recognizing both allogeneic cells and ovarian cancer cellscan be generated in vitro by using PBMC from patients (363 and 399),transducing with MOv-y, and stimulating with PBMC from donor (269). Thegraph shows that transduced and non transduced cells released GM-CSF inresponse to donor 269 PBMC. In addition, transduced patient (363 and399) cells released significant amounts of GM-CSF in response to IGROV,indicating that these transduced T cells were allogeneic- andIGROV-reactive.

FIGS. 8A-8F shows the phenotypic results of the bulk population of Tcells from patient 399 anti-269 (A-C) and of patient 363 anti-269 (D-F),where 8C and 8F demonstrate that the T cells express the chimeric MOv-γreceptor.

FIG. 9 shows the functional assay results from bulk MOv-γ transduced Tcells of patient 410, where T cells are both allo- and IGROV-reactive asmeasured by GM-CSF release (pg/ml).

FIGS. 10A-B show the phenotypic results of bulk MOv-y transduced T cellsof patient 410 (anti-556 donor), where the T cells are primarily CD4+and do in fact express the chimeric MOv-γ receptor.

FIGS. 11A-11J show the phenotypic results of individual daughter clonesfrom the bulk MOv-γ transduced T cells of patient 410 (anti-556 donor).FIGS. 11A-11E show that the T cells express the chimeric MOv-γ receptor,as demonstrated by the shift in peak on the X-axis. FIGS. 11F-11Jdemonstrate by FACS whether the clone is CD4 or CD8.

FIGS. 12A-12D show growth curve analysis of responder cells transducedwith dual specific T cells following stimulation with various allogeneiccell types (PBMC, DC, or B cells) and control at varyingstimulator:responder ratios.

FIGS. 13A-13D show growth curve analysis of responder cells transducedwith dual specific T cells following restimulation with variousallogeneic cell types (PBMC, DC, or B cells) and control at varyingstimulator:responder ratios.

FIG. 14 shows growth curve analysis of alloreactive T cells in MLR withvarious concentrations of IL-2.

FIG. 15 shows the MFG-MOv-γ-I-N retroviral vector.

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of a more complete understanding of the invention, thefollowing definitions are described herein. Nucleic acid sequencesinclude, but are not limited to, DNA, RNA or cDNA. Substantiallyhomologous as used herein refers to substantial correspondence betweenthe nucleic acid sequence for the V-J or V-D-J junctional sequences forthe α and β chains of the tumor antigen specific T-cell receptorsprovided herein and that of any other nucleic acid sequence. By way ofexample, substantially homologous means about 50-100% homology,preferably by about 70-100% homology, and most preferably about 90-100%homology between the nucleic acid sequences and that of any othernucleic acid sequence. In addition, substantially homologous as usedherein also refers to substantial correspondence between the amino acidsequence of the V-J or V-D-J junctional sequences of the antigenspecific T-cell receptors provided herein and that of any other aminoacid sequence.

Major Histocompatibility Complex (MHC) is a generic designation meant toencompass the histo-compatibility antigen systems described in differentspecies including the human leukocyte antigens (HLA). The term cancerincludes but is not limited to, melanoma, epithelial cell derivedcancers, lung cancer, colon cancer, ovarian cancer, breast cancer,kidney cancer, prostate cancer, brain cancer, or sarcomas.

The term melanoma includes, but is not limited to, melanomas, metastaticmelanomas, melanomas derived from either melanocytes or melanocyterelated nevus cells, melanocarcinomas, melanoepitheliomas,melanosarcomas, melanoma in situ, superficial spreading melanoma,nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma,invasive melanoma or familial atypical mole and melanoma (FAM-M)syndrome. The aforementioned cancers can be treated, assessed ordiagnosed by methods described in the present application.

The lymphocytes of the present invention are pre-selected for TCRshaving reactivity with specific antigens. These antigens are preferablystrong antigens. The term “strong antigen” as it is referred to hereinrelates to an antigen capable of inducing proliferation of pre-selectedadoptively transferred T cells. Examples of such antigens include butare not limited to alloantigens, viral agents and other foreign agents.Allogeneic agents or “alloantigens” are antigens derived fromgenetically non-identical members of the same species. Allogeneictissues, cells, proteins, peptides, nucleic acids and/or other cellularcomponents may be used to select an individual or subpopulation oflymphocytes. Examples of viral agents are well-known in the art, andinclude, but are not limited to, Epstein Barr virus and the Flu-virusand proteins, peptides, nucleic acids and other cellular componentsderived therefrom. Examples of other strong antigens include foreignproteins such as serum proteins from other species including bovine.

“Dual specificity lymphocytes” as that phrase is used herein refers tolymphocytes capable of reacting with both a tumor antigen and apre-selected strong antigen. The tumor antigen reactivity may beconferred by genetically modifying lymphocytes with a chimeric T cellreceptor gene encoding a binding site for the tumor antigen. Tumorantigen reactivity may also be conferred by native TCR itself.Reactivity with the pre-selected strong antigen(s) is preferablyconferred by in vitro expansion of the isolated population oflymphocytes by specific T cell activation using one or more pre-selectedstrong antigens.

“Chimeric receptor gene” refers to any receptor gene encoding a proteincontaining an extracellular recognition/binding site and transmembraneand intracellular portions capable of translating the binding of aligand to the recognition site to specific intracellular activities.Preferably, the chimeric receptor gene encodes sequences for T-cellreceptors or parts thereof which recognize tumor associated antigensand/or function to translate extracellular/cytoplasmic signal tointracellular activities in T-cells. One example of such a chimericreceptor gene encodes a single chain variable region from a monoclonalantibody joined to the Fc receptor chain capable of mediating T-cellreceptor signal transduction. Another preferred chimeric receptorcomprises an antibody variable region joined to the cytoplasmic regionof CD28 from a T cell or a similar region which can provide a T cellwith co-stimulation signals.

Additional examples of immune cell trigger molecules are any one of theIL-2 receptor (IL-2R) p55 (α) or p75 (β) or γ chains, especially the p75and γ subunits which are responsible for signaling T cell and NKproliferation.

Further candidate receptor molecules for creation of scFv chimeras inaccordance with the present invention include the subunit chains of Fcreceptors. In the group of NK-stimulatory receptors, the most attractivecandidates are the γ- and CD16α-subunits of the low affinity receptorfor IgG, FcγRIII. Occupancy or cross-linking of FcγRIII (either byanti-CD16 or through immune complexes) activates NK cells for cytokineproduction, expression of surface molecules and cytolytic activity[Unkeless, J. C. et al., Annu. Rev. Immunol. 6:251-281 (1988); Ravetch,J. V. and Kinet, J. P. Annu. Rev. Immunol. 9:457-492 (1991)]. In NKcells, macrophages, and B and T cells, the FcγRIII appears as aheterooligomeric complex consisting of a ligand-binding a chainassociated with a disulfide-linked γ or zeta chain. The FcγRIIIAsignalling gamma chain [Wirthmuller, V. et al., J. Exp. Med.175:1381-1390 (1992)] serves also as part of the FcεRI complex, where itappears as a homodimer, is very similar to the CD3 zeta chain, and infact can form heterodimers with it in some cytolytic T lymphocytes (CTL)and NK cells [Orloff, D. G., et al. Nature (London) 347:189-191 (1990);Lanier, L. G., et al. J. Immunol. 146:1571-1576 (1991); Vivier, E., etal. J. Immunol. 147:4263-4270 (1991)]. Most recently prepared chimerasbetween these polypeptides and the CD4 [Romeo, C. and Seed, B. Cell64:1037-1046 (1991)], the CD8 [Irving, B. A. and Weiss, A. Cell64:891-901 (1991)], IL-2 receptor chain [Letourneur, F. and Klausner, R.D. Proc. Natl. Acad. Sci. USA 88:8905-8909 (1991)] or CD16 extracellulardomains, proved to be active in signaling T cell stimulation even in theabsence of other TCR/CD3 components.

In one embodiment, the chimeric receptor genes encode amino acidsequences which provide for the V-J or V-D-J junctional regions or partsthereof for the alpha and beta chains of the T-cell receptor whichrecognize tumor associated antigens. In general, the chimeric T-cellreceptors recognize or bind tumor associated antigens presented in thecontext of MHC Class I. Many different tumor associated antigens areknown to the skilled artisan. A tumor antigen can be defined as amolecule that can be used to target therapy against a tumor and includesthose antigens only found on tumor cells (i.e. tumor specific), thosewhich are expressed on tumor cells and on limited normal tissues, i.e.differentiation antigens (including cancer-testis antigens) and thosewhich are over-expressed on tumor cells compared to the expression on awide variety of normal tissues (i.e. over-expressed antigens). Examplesof over-expressed antigens include, but are not limited to, Folatebinding protein (FBP), Erb-B2, GD-2, HMW-MAA, G250, TAG-72, NY-ESO-1,carcino-embryonic antigen and alpha-fetoprotein. Differentiationantigens include, for example, Tyrosinase, MART-1, MAGE and gp100 ofmelanoma. Tumor-specific antigens include, for example, mutant Ras,mutant p53, mutant Erb-B2 of a wide variety of tumors including breastand colon. Any of these tumor antigens can serve as the binding agentfor the TCR or chimeric receptor. The choice of which antigen to targetis within the skill of the ordinary artisan, and is based upon thespecific tumor being targeted.

In one preferred embodiment, the tumor associated antigens recognized bythe receptors of this invention are melanoma antigens. By way ofexample, melanoma specific T-cell receptors may recognize melanomaantigens in the context of HLA-A2.1 or HLA-A1. Examples of melanomaantigens which are recognized by the chimeric receptors include, but arenot limited to, MART-1, or peptides thereof or gp-100 or peptidesthereof. In a preferred embodiment the chimeric receptor recognizes orbinds to the MART-1 peptide, in particular epitopes M9-1 (TTAEEAAGI),M9-2 (AAGIGILTV), M10-3 (EAAGIGILTV), and M10-4 (AAGIGILTVI) (shown insingle letter amino acid code) or gp-100 peptide epitopes.

The chimeric receptor is provided as a recombinant DNA moleculecomprising all or part of the T-cell receptor nucleic acid sequence anda vector. The nucleic acid sequences encoding the α and β chains of aT-cell receptor of the present invention may be placed in a singleexpression vector. Alternatively the α chain and the β chain may each beplaced in a separate expression vector. Expression vectors suitable foruse in the present invention may comprise at least one expressioncontrol element operationally linked to the nucleic acid sequence. Theexpression control elements are inserted in the vector to control andregulate the expression of the nucleic acid sequence. Examples ofexpression control elements include, but are not limited to, lac system,operator and promoter regions of phage lambda, yeast promoters andpromoters derived from polyoma, adenovirus, retrovirus, cytomegalovirus(CMV), SRα, MMLV, SV40 or housekeeping promoters such as phosphoglycerolkinase (PGK) and β actin. Additional preferred or required operationalelements include, but are not limited to, leader sequences, terminationcodons, polyadenylation signals and any other sequences necessary orpreferred for the appropriate transcription and subsequent translationof the nucleic acid sequence in the host system. It will be understoodby one skilled in the art that the correct combination of required orpreferred expression control elements will depend on the host systemchosen. It will further be understood that the expression vector maycontain additional elements necessary for the transfer and subsequentreplication of the expression vector containing the nucleic acidsequence in the host system. Examples of such elements include, but arenot limited to, origins of replication and selectable markers and longterminal repeats (LTR) and internal ribosomal entry site (IRES). Theexpression vector may also include a leader peptide sequence. It willfurther be understood by one skilled in the art that such vectors areeasily constructed using conventional methods [Ausubel et al., (1987) in“Current Protocols in Molecular Biology”, John Wiley and Sons, New York,N.Y.] or commercially available.

Alternatively, the chimeric receptor gene may comprise a first genesegment encoding the single chain Fv receptor (scFv) of a specificantibody, i.e., DNA sequences encoding the variable regions of the heavyand light chains (V_(H) and V_(L), respectively) of the specificantibody, linked by a flexible linker, and a second gene segment whichcomprises a DNA sequence encoding partially or entirely thetransmembrane and cytoplasmic, and optionally the extracellular, domainsof a lymphocyte-triggering molecule corresponding to a lymphocytereceptor or part thereof. Thus, the scFvR design may be advantageousover the two-chain version of the receptor. It requires the expressionof only one gene instead of the gene pair required for the cTCR, therebyproviding simpler construction and transfection.

The scFv domain may preferably be joined to the immune cell triggeringmolecule such that the scFv portion will be extracellular when thechimera is expressed. This is accomplished by joining the scFv either tothe very end of the transmembrane portion opposite the cytoplasmicdomain of the trigger molecule or by using a spacer which is either partof the endogenous extracellular portion of the triggering molecule orfrom other sources. The chimeric molecules of the present invention havethe ability to confer on the immune cells on which they are expressedMHC nonrestricted antibody-type specificity. Thus, a continuouspolypeptide of antigen binding and signal transducing properties can beproduced and utilized as a targeting receptor on immune cells. In vivo,cells expressing these genetically engineered chimeric receptors willhome to their target, will be stimulated by it to attract other effectorcells, or, by itself, will mediate specific destruction of the targetcells. In a preferred embodiment, the target cells are tumor cells andthe scFv domain is derived from an antibody specific to an epitopeexpressed on the tumor cells. It is expected that such anti-tumorcytolysis can also be independent of exogenous supply of IL-2, thusproviding a specific and safer means for adoptive immunotherapy.

Besides the specific receptor chains specifically mentioned herein, thesingle chain Fv chimeras can be made by joining the scFv domain with anyreceptor or co-receptor chain having a similar function to the disclosedmolecules, e.g., derived from granulocytes, B lymphocytes, mast cells,macrophages, etc. The distinguishing features of desirable immune celltrigger molecules comprise the ability to be expressed autonomously(i.e., as a single chain), the ability to be fused to an extracellulardomain such that the resultant chimera is expressed on the surface of animmune cell into which the corresponding gene was geneticallyintroduced, and the ability to take part in signal transduction programssecondary to encounter with a target ligand.

The construction options for the production of chimeric T-cell receptorgenes and their corresponding proteins can be found in U.S. Pat. No.5,830,755 and U.S. application Ser. No. 08/547,263, both of which areincorporated herein by reference in toto. In addition, Hwu et al (Can.Res. (1995) 55:3369-3373) and Wang et al (Nat. Med. (1998) 492:168)describe details of introducing a chimeric receptor gene into cells andtreating tumors therewith. These references are incorporated herein byreference.

Specific expansion and specific activation of the T cells containing thechimeric T-cell receptor gene are important parts of the presentinvention. In one embodiment, the specific expansion step amplifies anindividual or a subpopulation of T cells whose endogenous TCR isdirected to the strong antigen(s) used to expand the T cells. In thisway, T cells which react with the antigen(s) are selected out andamplified from a mixed population of T cells originally obtained fromthe patient. The expanded lymphocytes are transduced with a chimericreceptor gene. These pre-selected, transduced T cells are introducedinto a patient, and the patient is immunized with the strong antigen(s).This in vivo immunization step serves to activate the pre-selectedadoptively transferred T cells and to target the lymphocytes to thecancer antigen through the chimeric receptor.

In a preferred embodiment of the invention, patients undergoleukapheresis to obtain peripheral blood lymphocytes (PBL). Thelymphocytes are separated from other cells. Various methods ofseparation are known to the artisan and can be utilized. One preferredseparation technique employs centrifugation on a Ficoll cushion. Thepreferred host cells transformed with all or part of the chimericreceptor nucleic acid sequences may include JURKAT-cells, T-lymphocytes,peripheral blood cells such as peripheral blood lymphocytes (PBL) andperipheral blood mononuclear cells (PBMC), dendritic cells, monocytes,stem cells, natural killer (NK) cells or macrophages.

Candidate immune cells to be endowed with antibody specificity usingthis approach are: NK cells, lymphokine-activated killer cells (LAK),cytotoxic T cells, helper T cells, and the various subtypes of theabove. These cells can execute their authentic natural function and canserve, in addition, as carriers of foreign genes designated for genetherapy, and the chimeric receptor shall serve in this case to directthe cells to their target. This approach can be applied also toanti-idiotypic vaccination by using helper T cells expressing chimericreceptors made of Fv of antiidiotypic antibodies.

The cells are activated with one or more preselected antigens. Anystrong antigen, i.e. one that is capable of inducing proliferation ofthe adoptively transferred lymphocytes, may be utilized for theactivation/selection step. The lymphocytes are preferably exposed to thestrong antigen for greater than one hour in the case of proteins andvirus or for at least 24 hours, preferably, continuously, for allogeneiccells as strong antigens. The strong antigen is provided up to aconcentration of 1 millimolar when proteins, peptides or cellularcomponents are used or at a ratio of one dual specific T-cell to 1-1000infectious viral particles or between 1 and 100 allogeneic cells pereach dual-specific T-cell. A combination of several stimuli mayoptionally be included in the activation/selection mixture. Onepreferred embodiment utilizes a donor's PBLs as an allogeneic agent.When donor PBLs are used, selection is carried out by co-culture ofirradiated donor PBMC with patient PBMC at a ratio preferably with therange of 2:1 to 5:1.

These cells are transduced with a chimeric receptor gene. “Transduction”or introduction of foreign DNA into the immune cells may be carried outby any manner known in the art, such as, for example, microinjection,electroporation, transduction, retroviral transduction or transfectionusing DEAE-dextran, lipofection, calcium phosphate, particle bombardmentmediated gene transfer or direct injection of nucleic acid sequencesencoding the chimeric receptors or other procedures known to one skilledin the art [Sambrook et al. (1989) in “Molecular Cloning. A LaboratoryManual”, Cold Spring Harbor Press, Plainview, N.Y.]. One preferredmethod of transduction follows the method described in Hwu, et al.,(1993) J. Immunol. 150:4104-4115. One preferred method of transductionresuspends the lymphocyte preparation in a retroviral supernatant at aconcentration range of 1×10² to 1×10¹° per ml, more preferably at arange of 1×10⁴ to 1×10⁸, most preferably at a concentration of 1×10⁶.Transduction may preferably be followed by a selection step, such as forexample using an antibiotic selection marker on the chimeric receptorgene construct, such as the neomycin resistance gene.

The preselected transduced lymphocytes may be cultured for several days.Between days 14-21, it may be desirable to screen the cells for specificcytokine release against ovarian tumor antigens and/or assay forphenotype integrity. At this time, it may also be desirable torestimulate the lymphocyte population with the strong antigen.Restimulation using a strong antigen is preferably carried out at asimilar concentration as used for the initial stimulation for a similartime period. If donor cells are used as the allogeneic agent,restimulation is preferably carried out at a ratio of 0.5:1 to 4:1, morepreferably at a ratio of 1:1 to 2:1 (donor:patient). These cells can bedirectly reintroduced into the patient or can be frozen for future use,i.e. for subsequent administrations to this patient.

Upon expansion of lymphocytes in IL-2 containing media, patients receivepreselected transduced lymphocytes intravenously. Optionally, thepatient may also receive IL-2, preferably after the lymphocyte infusion.It is preferable that the IL-2 be provided at a dosage range of 1200IU/kg to 1,200,000 IU/kg, more preferably at 120,000 IU/kg every 12hours. After the first administration, patients may again receivepreselected, transduced lymphocytes intravenously with or withoutsimilar doses of IL-2. Dual specificity lymphocytes are administered ata dosage range of 1×10⁶ to 1×10¹⁵, more preferably 1×10⁸ to 1×10¹¹, mostpreferably 3×10⁹ to 5×10¹° cells. Further details on dosage andfrequency of cells are provided in U.S. Pat. No. 5,399,346, which isincorporated herein by reference, in toto.

The present invention provides a method of inhibiting or preventing thegrowth of tumor cells by exposing tumor cells to the dual specificitylymphocytes provided herein. The dual specificity lymphocytes may beused for either prophylactic or therapeutic purposes. When providedprophylactically, the dual specificity lymphocytes is provided inadvance of any evidence or symptom in the mammal due to cancer, inparticular, melanoma. The prophylactic use of the dual specificitylymphocytes serves to prevent or attenuate cancer, in particularmelanoma, in a mammal. When provided therapeutically, dual specificitylymphocytes are provided after the onset of the disease in the mammal.The therapeutic administration of the dual specificity lymphocytesserves to attenuate the disease.

Cell-based immunotherapy currently utilizes the adoptive transfer topatients of tumor specific TIL which are generically expanded ex vivo[Rosenberg S. A. 1992. J. Clin. Oncol., 10:80; Rosenberg S. A., et al.N. Engl. J. Med., 319:1676; Hwu P., et al. 1993. J. Exp. Med., 178:361].T-cell specificity may be redirected by combination of the in vitrotransfer of the nucleic acid sequences encoding the tumor associatedantigen specific T-cell receptors and selective expansion of thelymphocytes with one or more strong antigens. By way of example, aheterogenous population of T-cells, such as TIL, may be made moreeffective by conferring anti-tumor reactivity to non-specific T-cellpopulations within the TIL, and selective expansion of T lymphocytes toamplify T lymphocytes reactive with one or more pre-selected strongantigens.

Cells that can be modified to produce dual specificity lymphocytesinclude, but is not limited to, lymphocytes, cytotoxic T-lymphocytes,hematopoietic stem cells, monocytes, stem cells, peripheral blood andnatural killer cells. In a preferred embodiment, T-cells can begenetically modified to express the tumor antigen specific T-cellreceptors. Constructs containing all or parts of the nucleic acidsequences encoding the chimeric T-cell receptors may be introduced inT-lymphocytes by conventional methodology. By way of example suchmethods include, but are not limited to, calcium phosphate transfection,electroporations, lipofections, transduction by retroviruses, injectionof DNA, particle bombardment and mediated gene transfer use of aretroviral vector, viral vectors, transduction by viral co-culturingwith a producer cell line. Preferably, the construct or constructscarrying the nucleic acid sequences encoding the chimeric T-cellreceptors are introduced into the T-cells by transduction with viralsupernatant or co-cultivation with a retroviral producer cell line.Examples of vectors that may be used include, but are not limited to,defective retroviral vectors, adenoviral vectors, vaccinia viralvectors, fowl pox viral vectors, or other viral vectors [Mulligan, R.C., (1993) Science 260:926-932]. Eukaryotic expression vectors G1EN[Treisman, J., et al., Blood, 85:139; Morgan et al. (1992) Nucleic AcidsRes. 20:1293-1299], LXSN [Miller, A. D., et al. Methods Enzymol.,217:581-599 (1993); Miller, A. D., et al. BioTechniques, 7:980-988(1989); Miller, A. D., et al., Mol. Cell Biol., 6:2895-2902 (1986);Miller, A. D. Curr. Top. Microbiol. Immunol., 158:1-24 (1992)], andSAM-EN [Treisman, J., et al., Blood, 85:139] may also be used.Individual constructs carrying the genes coding for the alpha and betachains that comprise the receptor may be introduced into theT-lymphocytes or alternatively, an individual construct carrying thenucleic acid sequences encoding for both the α and β chains of theT-cell receptor may be in a single construct. Preferably, a retroviralvector, for example a vector with the murine moloney leukemia viral LTRpromoting transcription of the T-cells receptor genes is used. In apreferred embodiment non-replicating retroviral vectors are used.Alternatively, the genes can be expressed using an internal housekeepingpromoter, such as that from the phosphoglycerol kinase (PGK) gene.

The α and β chains of the T-cell receptor may either be expressed onseparate retroviral vectors, or on the same retroviral vector, separatedby an internal ribosomal entry site (IRES) [Treisman, J., et al., Blood,85:139; Morgan, R. A., et al., Nucleic Acids. Res., 20:1293-1299(1992)]. Using an IRES-containing vector, allows both T-cell receptorgenes to be translated from a single RNA message. Examples of whereT-lymphocytes can be isolated, include but are not limited to,peripheral blood cell lymphocytes (PBL), lymph nodes, or tumorinfiltrating lymphocytes (TIL), or blood. Such lymphocytes can beisolated from the individual to be treated or from a donor by methodsknown in the art and cultured in vitro [Kawakami, Y. et al. (1989) J.Immunol. 142: 2453-3461]. Alternatively, a single chain Fv receptor genemay be constructed and used as the chimeric receptor gene.

The T-cells may be incubated with a retroviral producer cell linecarrying retroviral expression vectors or with viral supernatant.Viability of the lymphocytes may be assessed by conventional methods,such as trypan blue dye exclusion assay. The genetically modifiedlymphocytes expressing the desired melanoma specific T-cell receptor maythen be administered to a mammal, preferably a human, in need of suchtreatment in a therapeutically effective amount. The dosing regimes orranges of lymphocytes used in the conventional tumor infiltratinglymphocyte (TIL) therapy [Rosenberg, et al. (1994) J. Natl. Canc. Inst.,Vol. 86:1159] may be used as general guidelines for the doses or numberof T-lymphocytes to be administered to mammal in need of such treatment.By way of example, a range of about 1×10¹⁰ to about 1×10¹¹ T-cells foreach cycle of therapy may be administered in the methods providedherein. Examples of how these antigen specific T-cells can beadministered to the mammal include but are not limited to,intravenously, intraperitoneally or intralesionally. Parameters that maybe assessed to determine the efficacy of these transduced T-lymphocytesinclude, but are not limited to, production of immune cells in themammal being treated or tumor regression. Conventional methods are usedto assess these parameters. Such treatment can be given in conjunctionwith cytokines or gene modified cells [Rosenberg, S. A. et al. (1992)Human Gene Therapy, 3: 75-90; Rosenberg, S. A. et al. (1992) Human GeneTherapy, 3: 57-73] chemotherapy or active immunization therapies. One ofskill in the art will appreciate that the exact treatment schedule anddosages, or amount of T-lymphocytes to be administered may need to beoptimized for a given individual.

This invention also relates to pharmacological compositions comprisingthe dual specificity lymphocytes. The formulations of the presentinvention, both for veterinary and for human use, comprise eachcomponent individually or as a composition as described above, togetherwith one or more pharmaceutically acceptable carriers and, optionally,other therapeutic ingredients. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any method known in the pharmaceutical art.

Preparation of the Pharmaceutical Compositions Include the step ofbringing into association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product intothe desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1-2.0M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharide,amino acids, inorganic acids, and organic acids which may be used eitheron their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11-10,000 parts by weight per part byweight of each component or the composition. If two or more stabilizersare to be used, their total amount is preferably within the rangespecified above. These stabilizers are used in aqueous solutions at theappropriate concentration and pH. The specific osmotic pressure of suchaqueous solutions is generally in the range of 0.1-3.0 osmoles,preferably in the range of 0.8-1.2. The pH of the aqueous solution isadjusted to be within the range of 5.0-9.0, preferably within the rangeof 6-8. In formulating each component separately or as a composition ofthe present invention, anti-adsorption agent may be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the cells or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate the9-cis-retinoic acid or derivatives thereof alone or in combination withantineoplastic agents thereof into particles of a polymeric materialsuch as polyesters, polyamino acids, hydrogels, poly(lactic acid) orethylene vinylacetate copolymers. Alternatively, instead ofincorporating these agents into polymeric particles, it is possible toentrap these materials in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the component may be combined withtypical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

The administration of the compositions or of each individual componentof the present invention may be for either a prophylactic or therapeuticpurpose. The methods and compositions used herein may be used alone inprophylactic or therapeutic uses or in conjunction with additionaltherapies known to those skilled in the art in the prevention ortreatment of cancer. Alternatively the methods and compositionsdescribed herein may be used as adjunct therapy. Veterinary uses arealso intended to be encompassed by this invention.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Ineffective Treatment of Cancer Patients

Previously, chimeric receptors against ovarian cancer (MOv-γ) were foundto be functional in primary T cells in vitro and in vivo [Hwu, P., etal. J. Exp. Med., 178:361-366, 1993; Hwu, P., et al., Cancer Res.,55:3369-3373, 1995]. The effects of treating eight patients withadvanced ovarian cancer with T cells transduced with chimeric receptorgenes derived from a monoclonal antibody against ovarian cancer, MOv-18alone, and without any specificity were observed. Tumor-infiltratinglymphocytes (TIL) or anti-CD3 stimulated peripheral blood lymphocytes(PBL) retrovirally transduced with the MOv-18 chimeric receptor gene(MOv-7) were generated in large numbers of MOv-PBL which remained highlyreactive against ovarian cancer cells in vitro prior to infusion.Patients were treated with up to 5×10¹° transduced PBL for CD3 incombination with systemic IL-2 (120,000 CU/kg). The results of thisclinical trial demonstrated that cells were directed to lung, liver, andspleen, but did not specifically localize at tumor sites. Despitespecific in vitro reactivity of MOv-PBL against ovarian cancer cells,none of the patients responded to the lymphocyte infusion. The number ofcirculating transduced cells following MOv-PBL infusion was determined.Between 4-6 days following MOv-PBL infusion, between 0.01 and 1%circulating transduced cells were detected; however, between 12-31 days,the majority of transduced cells were undetectable. FIG. 2 shows thepercentage of circulating transduced cells during the course of thetreatment after MOv-PBL infusion.

Example 2 Functionality of MOv-PBL after Ineffective Treatment of CancerPatients

In order to address the question as to why patients transduced withchimeric receptor genes did not respond to treatment although 10% oftransduced cells were found circulating in one patient's PBMC analyzedafter 5 days post MOv-PBL infusion, the functionality of MOv-PBL aftercell transfer was determined. Fresh uncultured PBMC from the day 5 postMOv-PBL infusion time point were co-cultured with ovarian cancer cellsor melanoma cells (888 mel or 1300 mel). Supernatants were assayed forIFN-γ by enzyme-linked immunosorbent assay (ELISA) and lysates wereanalyzed for IFN-γ mRNA using Taqman. No significant IFN-γ release wasseen using the fresh day 5 PBMC. To reisolate the adoptively transferredMOv-PBL, the PBMC from day 5 were cultured in G418 (for neomycinresistant selection), anti-CD3, and IL-2. After 17 days, the culture washighly enriched for reisolated MOv-PBL (69% positive for gene) that werecapable of specifically producing large amounts of cytokine in responseto ovarian tumor cells. As measured by IFN-γ release (pg/ml), Table 1shows that MOv-PBL retained their ability to recognize tumor afteradoptive transfer. The NV PBL group did not result in IGROV or melanomareactivity. The NV PBL and G418 group also did not result in significantIGROV, 888 mel, or 1300 mel reactivity. Both cultured MOv-PBL and thereisolated PBL were significantly IGROV-reactive. These results indicatethat MOv-PBL retained their ability to recognize tumor after adoptivetransfer, although culturing was necessary to observe anti-tumorreactivity.

TABLE 1 IFN-γ Release (pg/ml) GROUP IGROV 888 mel 1300 mel NV PBL 0 0 0NV PBL + G418 0 49 0 MOv-γ transduced 3582 3 14 PBL Reisolated PBL 29120 192

Example 3 In Vivo Expansion of Alloreactive Cultured T Cells in MurineModels

In order to determine the effects of allogeneic immunization ofadoptively transferred T cells, anti-allogeneic mouse (C57BL/6) T cellswere raised in a mixed lymphocyte reaction (MLR) for 7 days andrestimulated for 6 additional days. Thy 1.1+ alloreactive (H2-banti-H2-d) T cells were generated and expanded and 1×10⁷ cells wereadoptively transferred into congenic C57BL/6 mice (Thy 1.2) byintravenous injection. The C57BL/6 mice were immunized with allogeneicantigen presenting cells from BALB/c mice on days 2, 5, and 8 aftertransfer. Where stimulators, for example, either allogeneic splenocytesor allogeneic dendritic cells (DC), were used to immunize the mice.Stimulators are strong antigenic agents which activate responder cells,for example lymphocytes. There were 3 mice per condition group. Tissues(spleen, lung, and blood) were harvested on day 11 and Thy 1.1 cellswere quantified following staining and fluorescence-activated cellsorter (FACS) analysis. The percent of Thy 1.1 cells in tissue wasmeasured by comparing those immunized with allogeneic splenocytes andallogeneic DC. Adoptively transferred, cultured alloreactive T cellswere found to expand in vivo following immunization with allogeneicantigen presenting cells and increased the survival of invitro-cultured, adoptively transferred allo-reactive T cells uponimmunization with allogeneic cells (FIGS. 3A and 3B).

Example 4 Generation of Dual Specificity Mouse T Cells

C57BL/6 mice T cells (2×10⁷) were raised in a mixed leukocyte reaction(MLR) in 24 well plates and 10 CU/ml IL-2 with restimulation on day 7.G418 (0.5 mg/ml) was added for 6 days until day 13 and assayed forMOv-'y expression and IFN-γ secretion in response to various targetcells. The results of Table 2 and FIG. 4 show that dual specificity Tcells expressing chimeric MOv-γ with significant anti-allogeneic andanti-FBP activity as measured by IFN-γ (pg/ml) was generated.

TABLE 2 Non-transduced T Media cells MOv-γ T cells Media 0 0 0 CT26(allogeneic 0 65,200 138,700 H-2d) 24JK (H-2b) 0 0 0 24JK-FBP 0 0 5200888 mel 0 0 0 IGROV (ovarian 0 0 37,500 FBP+)

Example 5 Protection Against Tumor Challenge by Adoptive Transfer ofDual Specificity T Cells

C57BL/6 mice received 1×10⁷ dual specificity allogeneic/MOv-γ T cellsfollowed by subcutaneous immunization with 5×10⁷ allogeneic splenocytesfrom donor mice 2 days later. Seven days after immunization, mice werechallenged with 2×10⁵ 24JK-FBP ovarian cancer tumor cellssubcutaneously. The 24JK is a clone from the3-methylcholanthrene-induced, poorly immunogenic MCA 102 murine sarcoma,where 24JK-FBP is 24JK transduced with the folate binding protein (FBP)gene, which is expressed highly on ovarian adenocarcinomas and MOv-γ isa mAb that binds FBP. Five mice per condition were then monitored for 20days. FIG. 5 shows that in vivo immunization with allogeneic splenocytesfrom donor mice, in combination with administration of dual specificityT cells protected mice much more significantly than T cells alone.Specifically, the combined conditions result in 100% tumor-free micewhile mice infused with dual specificity T cells alone resulted in 25%tumor-free mice.

Example 6 Treatment of Established Subcutaneous Tumor in ImmunodeficientMice with Adoptive Transfer of Dual Specificity T Cells Followed byImmunization

In order to determine whether the combination of adoptively transferreddual specificity T cells and immunization with allogeneic cells caninhibit established subcutaneous tumors, 3 RAG-1 immunodeficient knockout mice per condition were injected on day 0 with 2×10⁵ tumor cells,specifically, 24JK and 24JK-FBP tumor cells. On day 3, mice receivedeither 1×10⁷ dual specificity T cells, 1×10⁷ non-transduced (NV) Tcells, or no treatment. Subcutaneous immunization with 5×10⁷ allogeneicsplenocytes was performed on days 5, 8, and 11. On day 15, one mouse pergroup having a middle sized tumor was sacrificed in order to determinethe effect of immunization on transferred T cell numbers. Dualspecificity T cells inhibited the tumor and this effect was augmented byimmunization. FIG. 6 shows that mice injected with 24JK-FBP tumor cellsfollowed by transduced dual specificity T cells and immunization orboost, resulted in the smallest tumor size throughout the time course of29 days.

Example 7 Generating Dual Specificity Human T Cells

Since murine studies indicated that dual specificity T cells can begenerated and functional, used for prevention, and treatment of tumors,dual specificity human T cells were generated. On day 0, 2×10⁶ respondercells or peripheral blood mononuclear cells (PBMC) from patients 363 and399 were cultured in the presence of 2×10⁵ irradiated (5 Krads)stimulator cells or PBMC from donor 269 [Aim V medium (LifeTechnologies)/5% human serum, type AB (Valley Biomedical); 100 CUIL-2/ml] per well in a 24 well plate. On day 12 patient cells wererestimulated with donor 269 PBMC (5×10⁵ T cells and 2×10⁵ irradiatedstimulators/well). On days 15 and 16, cultured cells were transducedwith MOv-'y supernatant during centrifugation at 1000 g (2700 rpm inSorvall tabletop centrifuge for 1 hour) in the presence of 8 μg/mlpolybrene, a polycation which aids in retroviral infection. On days18-22 cells were incubated with 0.5 mg/ml G418 (Geneticin; LifeTechnologies) per day for neomycin selection. On day 27 cells wererestimulated with donor 269 PBMC as above. On day 35, the bulkpopulation was assayed and cloned at 1 cell/well using standard surgerybranch (SB) method with OKT3 stimulation and PBMC from another donor.Results indicated that human anti-allogeneic T cells raised from PBMC363 and 399 against PBMC269 are both allogeneic-reactive andMOv-reactive following transduction with chimeric MOv-γ (FIG. 7). Ondays 49-52, the clones were characterized. The anti-allogeneicreactivity of PBMC 399 clones as measured by GM-CSF release (pg/ml)indicated that 62.5% of the clones were allo-reactive, 23.5% of the top17 allo-reactive clones were demonstrated to be IGROV-reactive, and 12%were both allo-reactive and IGROV-reactive. The anti-allogeneicreactivity of PBMC 363 clones as measured by GM-CSF release indicatedthat 58% of the clones were alto-reactive, 57% of the top 7allo-reactive clones were IGROV-reactive, and 36% were both allogeneic-and IGROV-reactive. Phenotypic characterization of the bulk populationis described by FACS analysis (FIGS. 8A-8F), where FIGS. 8A-8C representthe phenotype for the bulk population of patient 399 anti-269 and FIGS.8D-8F represent the phenotype for the bulk population of patient 363anti-269. FIGS. 8A and 8D show the control results using non-specificIgG2a; FIGS. 8B and 8E describe the CD4 and/or CD8 phenotype of thepopulation of cells; and FIGS. 8C and 8F show the shift of the MOvantibody-stained cells which represents the presence of the ovariancancer (MOv)-specific receptor on T cells.

Example 8 Generating Dual Specificity T Cells and Expanding Using the SBREP Method

In order to determine whether or not individual T cells were bothallogeneic- and MOv-γ-reactive, PBMC from patient 410 were used togenerate and characterize dual specificity T cells. On day 0, 5×10⁷ PBMCfrom 410 were cultured in the presence of 2×10⁶ responders irradiatedallogeneic PBMC/well from donor 556 (AIM-V/5% human serum; 100 CUIL-2/ml; in 24 well plates). On days 6 and 8, PBMC from 410 weretransduced with MOv-y supernatant supplemented with 8 μg/ml polybrene.Plates were centrifuged at 1000 g or 2700 rpm. On days 9-13, G418selection was performed using 0.5 mg/ml per day. On day 14, the bulkpopulation was assayed and cloned at a 1 cell/well ratio for 40 platesusing standard SB method with PBMC from random donor (5×10⁴/well); OKT3(30 ng/ml); and IL-2 (100 CU/ml). On days 29-31, the daughter cellclones were assayed against allogeneic and ovarian cancer targets, where54% of 150 clones were allo-reactive and 67% of 34 clones were folatebinding protein (FBP)-reactive. Therefore, 36% of the clones were shownto be reactive with both allogeneic PBMC and IGROV. On day 32, ten dualspecificity clones were expanded using random allogeneic PBMC and the SBRapid Expansion Protocol (REP). On days 43-44, the clones were retested.The ten most reactive MOv-γ transduced T cells (410) were tested andfound to be both allo- and IGROV-reactive as measured by GM-CSF release(pg/ml). The results of FIG. 9 show that non-transduced T cells areallogeneic-reactive, whereas, bulk MOV-γ transduced 410 T cells are bothallogeneic- and IGROV-reactive as measured by GM-CSF release (pg/ml).FIGS. 10A-10B and Table 3 describe the phenotypic characteristic of thebulk population of patient 410 anti-556 donor cells. The majority ofcells are CD4 helper T cells and were found to be reactive againstIGROV. Therefore, dual specificity human T cells can be grown torecognize both allogeneic targets as well as tumor. Table 4 shows GM-CSFsecretion from ten of the most reactive transduced dual specificity PBMC410 clones. These results confirm that each clone is both alloreactiveas demonstrated by the high GM-CSF release in response to PBMC 556 donorand specific for ovarian cancer as demonstrated by the high GM-CSFsecretion in response to IGROV. FIGS. 11A-11J show the phenotypecharacterizations of representative selected clones 1, 3, 5, 8, and 9.Although there is some variation in whether the clone is CD4+, CD8+, orboth CD4+/CD8+, all clones recognize the presence of the ovarian cancerspecific receptor on T cells.

TABLE 3 Quad Events % Gated % Total CD4+/CD8− 6237 82.27 43.42 CD4+/CD8+233 3.07 1.62 CD4−/CD8− 116 1.53 0.81 CD4−/CD8+ 995 13.12 6.93

TABLE 4 CLONE 1 2 3 4 5 6 7 8 9 10 PBMC410 1010 650 1790 640 1580 23802330 70 250 0 PBMC556 2300 2410 >5000 3200 >5000 4870 >5000 3050 13301020 888 105 0 20 0 750 130 100 0 0 10 mel IGROV 2820 2690 2770 22003440 4850 2380 2290 5270 2400

Example 9 Optimization for Generating the Highest Number of DualReactive T Cells

In order to maximize proliferation and reactivity against bothallogeneic and tumor targets which can be utilized for patienttreatment, the type of stimulator cells, the stimulator:responder ratioin MLR, IL-2 concentration, and conditions for restimulation must beoptimized. Therefore, 2×10⁶ fresh responder PBMC from a normal donorwere incubated in wells of a 24 well plate with a stimulator comprisingone of irradiated allogeneic PBMC, B cells, or DC at the followingstimulator to responder ratios: 5:1, 1.5:1, 1:2, 1:6, 1:20, and 1:60 inAIM-V/5% human serum; 100 CU IL-2/ml). After 3 days incubation, thecells were transduced with MOv-γ/TCR by replacing of the media withretroviral supernatant followed by centrifugation at 2700 rpm for 1hour. Transduction was repeated the following day, and cells were thenselected in 0.5 mg/ml G418. FIGS. 12A-12D show growth curves followingstimulation with various allogeneic cell types and at variousstimulator:responder ratios. FIG. 12D is the control responder withoutstimulators. Table 5 describes the phenotype of T cell culturesgenerated from a variety of APC and stimulator to responder ratios onday 17.

Tables 6-9 show the results of functional assays of cells on day 17after stimulation for the various APC and stimulator: responder ratios,where the different ratios were performed in duplicates. Allogeneic PBMCwere determined to be good stimulators for MLR, and result in highlevels of expansion when using stimulator:responder ratios ranging from1.5:1 to 5:1. Functional assays demonstrated that transduced cellsgenerated from PBMC stimulators were capable of recognizing allogeneicand tumor targets.

TABLE 5 Phenotype CD4+/ APC Stim:Resp CD3+ CD4+ CD8+ CD4+/CD8+ CD8− PBMC 1:60 93  4 51 2 43  1:20 94  4 49 2 45 1:6 1:2 88  9 43 4 45 1.5:11  9115 41 5 40 5:1 95 19 42 7 32 DC  1:60 96  9 54 5 33  1:20 99 25 44 10 21 1:6 100  64 11 20   5 1:2 ND ND ND ND ND 1.5:1   ND ND ND ND ND 5:1ND ND ND ND ND B-cells  1:60 90 10 41 4 45  1:20 86 18 34 6 42 1:6 88 2528 8 40 1:2 96 54 16 12  18 1.5:1   98 67 12 15   7 5:1 99 76 10 11   3OKT3 — 99 56 27 16   2 No — 93  5 52 3 40 OKT3 (ND = Not done) (CD4/CD8double negative cells were CD3+, CD19−, CD14−, CD11c−)

TABLE 6 PBMC (IFN gamma pg/ml) Stim:resp 1:60 1:20 1:6 1:2 1.5:1 5:1media  59  48  37  31  33  38  27  24  44  34  50  49 alone IGROV-12785  2200  3122  2973  3320  2815  2319  2349  3251 3755 4733  4279 888185 614 165 172  65  66  38  42  65  69 100  91 stim. 107  97 174 261443 631 548 612 1553 1693 1802  1244 PBMC responder 245 156 212 176 131136 313 118  141  148 269  172 PBMC coated 7018  7155  5916  6172  4950 6083  3627  3765  5078 5019 >11060   7391 OKT3

TABLE 7 DC (IFN gamma pg/ml) Stim:resp 1:60 1:20 1:6 media alone  28  34 27  27  28  60 IGROV-1 2636  3102 3785 4131 1583  889 888  79  74  471 422  51  62 stim. PBMC 995 1054 1683 1463 1553 1921 responder 350  121 76  85  104  57 PBMC coated OKT3 5965  7019 9385 9727 6211 5108

TABLE 8 B Cells (IFN gamma pg/ml) Stim:resp 1:60 1:20 1:6 1:2 1.5:1 5:1media  30  35  30  41  81 62 39 28 10 10  7  2 alone IGROV-1 1374 15432894 3270 4634  3548  3686  3181  1583  1463  350  341 888  124  122  79 63 100 95 47 45 11 11  2 3 stim.  680 1090 1433 1314 1081  895  761 518  308  228  96 123 PBMC responder  139  114  399  161 349 133  83 7950 44 31  33 PBMC coated 7096 4999 5640 4871 6240  7303  6014  5916 5384  5728  886  994 OKT3

TABLE 9 Stimulators (IFN gamma) +OKT3 No OKT3 media alone 2 0 23 23IGROV-1 1832  2259  2646  2587  888 16 18 50 84 stim. PBMC 16 17 43 72responder 90 50 189  131  PBMC coated OKT3 3656  3815  6585  5561 

Following the first stimulation, 4×10⁵ of the T cells from each groupwere added to the appropriate number and type of stimulator cells (fromthe same allogeneic donor) in a 24 well plate with 50 CU IL-2/ml and 5%AIM-V human serum. IL-2 (50 CU/ml) was added every 2 days. FIGS. 13A-13Dshow the number of transduced cells upon further stimulation.

Example 10 Optimization of IL-2 Concentrations for Maximized Cell Numberand Reactivity Against Allogeneic and Tumor Targets

Human alloreactive T cells from cryopreserved cells were generated usingPBMC at a ratio of 1:1 (2×10⁶ stimulator to 2×10⁶ responder/well), inthe presence of various IL-2 concentrations. T cells were transducedwith MOv-γ and selected in 0.5 mg/ml G418 for 5 days. Proliferation andreactivity against allogeneic and tumor targets were determined. FIG. 14shows T cell growth in MLR with various IL-2 concentrations in CU/ml.Results indicate that as the concentration of IL-2 increases, the numberof T cells generated also increases. The phenotypic characteristics of Tcell cultures generated from a variety of IL-2 concentrations isdepicted in Table 10. At all concentrations of IL-2, T cells arepredominantly CD3-1- and then CD4+. In Table 11, the function of MOv-γtransduced alloreactive T cells grown in a variety of IL-2concentrations as measured by IFN-γ release (pg/ml) demonstrates that 50CU IL-2/ml provides good expansion, as well as a high level ofreactivity against both allogeneic and tumor targets.

TABLE 10 [IL-2] Phenotype (CU/ml) CD3+ CD4+ CD8+ CD4+/CD8+ CD4+/CD8−12.5 80 50 23 4 22 25 83 58 22 7 14 50 71 49 22 2 27 100 70 38 28 6 28

TABLE 11 Stimulator (IFN gamma pg/ml) media Responder Stimulator alone888 mel IGROV-1 PBMC PBMC media alone 0 0 0 0 0 0 0 0 0 0 12.5 CU/mlIL-2   4 0 353 318 879 725 278 263 3900 3850 25 CU/ml IL-2 4 4 118 1382280 2140 239 210 2420 3450 50 CU/ml IL-2 2 12 68 77 2460 2210 235 2484880 4830 100 CU/ml IL-2  8 8 746 688 2900 2900 231 247 1850 2830

Example 11 Generation of Human Peripheral Blood Lymphocytes Transducedwith the Mov-γ Chimeric Receptor Gene

Serum-free AIM-V medium is supplemented with penicillin G (50 units/ml),and L-glutamine (292-584 mg/ml, 2 mM), as well as IL-2 (100 CU/ml). Ifnecessary, AIM-V medium can also be supplemented with 1-10% human serum(type AB heat inactivated at 56° C. for 30 minutes).

PBL is isolated by leukapheresis. Lymphocytes are separated bycentrifugation 1000 g (2700 rpm) on a Ficoll cushion. PBL is subjectedto multiple exposures of retroviral supernatant, for up to 3 days. ThePBL is then selected for 5 days in 0.5 mg/ml of the neomycin analog G418(Geneticin; Gibco; Grand Island, N.Y.). Following G418 selection, PBL isexpanded in AIM V media with 100 CU/ml IL-2. If necessary, AIM V issupplemented with 1-10% human AB serum. The exact days stated below isan approximation of what is expected for PBL transduction.

On Day 0, isolate PBMC from the leukapheresis preparation from patientand donor by Ficoll-Hypaque gradient centrifugation. Wash in Ca²⁺-,Mg²⁺-, Phenol red free Hanks' balanced salt solution (HBSS;BioWhittaker), then resuspend in AIM V medium supplemented with 50 CU/mlof IL-2. Irradiate donor PBMC with 5000 rads. Co-culture irradiateddonor PBMC with patient PBMC at a ratio of 2:1 to 5:1.

On Day 3, harvest PBMC and resuspend in retroviral supernatantsupplemented with 50 CU/ml of IL-2 and 8 μg/ml polybrene. Replate PBMCat a concentration of 1×10⁶ per ml; 2 ml per well in 24 well plates.Centrifuge plates at 1000 g (2700 rpm in Sorvall tabletop centrifuge)for 1 hour.

On Day 4, remove 1 ml of media per well and replace with 1 ml of freshlythawed retroviral supernatant supplemented with 50 CU/ml of IL-2 and 8μg/ml polybrene. Centrifuge plates at 1000 g (2700 rpm in Sorvalltabletop centrifuge) for 1 hour.

On Day 5, remove two-thirds of media per well and replace with AIM Vmedium supplemented with 50 CU/ml of IL-2.

On Day 6, harvest the PBL, and resuspend at 1×10⁶ per ml in AIM V mediumsupplemented with 50 CU/ml of IL-2 and 0.5 mg/ml G418. Replate cells inappropriate size Fenwal bag (Baxter) or T-175 tissue culture flask.Aliquot 5×10⁶ cells from non-transduced (NV) and transduced PBL groupsfor PCR analysis.

Every 2-3 days, count cells and dilute to 1×10⁶ cells per ml in AIM Vmedium supplemented with 50 CU/ml of IL-2 and 0.5 mg/ml G418.

On day 11, harvest the PBL, and resuspend at 1.5×10⁶ per ml in AIM Vmedium supplemented with 50 CU/ml of IL-2. Replate cells in appropriatesize Fenwal bag or T-175 tissue culture flask.

Between Days 14-21, the PSL are screened for specific cytokine releaseagainst the ovarian tumor cell line IGROV-1, and phenotype by FACSanalysis. Aliquot 5×10⁶ transduced, selected PBL for PCR analysis. Sendsamples for S+L-assay for retrovirus.

On approximately day 21, the patient PBMC are restimulated withirradiated donor PBMC at a ratio of 1:1 to 2:1 (donor:patient).

The density of PBL is maintained between 1×10⁶ and 2.5×10⁶ PBL/ml. OncePBL have begun to grow, the cultures are assessed for growth every 3-4days to insure that they do not increase beyond 2.5×10⁶/ml.

Once the total PBL count reaches about 5×10⁸, PBL are removed from thetissue culture plates or flasks and cultured further in Fenwal PL732cell culture bags. These bags have a 1-liter capacity, but normally 500mls medium are the maximum used in each bag. PL732 bags are gaspermeable, but impermeable to fluids. Thus, oxygen and CO₂ are freelyexchanged while tissue culture medium and cells are maintained inside.Using an inverted syringe (plunger removed), suspended by a clamp on aring stand, connect the needle-adapter end of the syringe to thefemale-luer port of the 1 liter PL732 bag. Pour the cells from the 250ml conical centrifuge tube into the bag through the syringe, and toobtain a cell concentration of 1×10⁶ PBL/ml add the appropriate volumeof fresh AIM-V (serum-free medium) containing the following addedsupplements (final concentrations): 50 units/ml penicillin G sodium(BioWhittaker), 146 g/ml L-glutamine (Media Tech), 1.25 mg/ml Fungizone,and IL-2 (50 CU/ml).

As the PBL continue to grow, they are transferred to a 3 liter capacityPL732 bag (1500 ml/bag, 1.5×10⁹ PBL/bag) via the male- and female-luersterile tubing ports of both the 1 liter and 3 liter bags. Theappropriate volume of AIM V medium is then added to maintain the PBL at1×10⁶/ml. Medium is added to the 3 liter bag using the same steriletubing attached to an inverted syringe, as described above. PL732 bagsare advantageous in that access to the medium containing cells islimited to injection sites and sterile tubing ports, both of which canbe maintained aseptically.

After the PBL are transferred to PL732 bags, PBL cell counts are doneevery 3-4 days. When the PBL density reaches 2.0×10⁶/ml or greater, thePL732 bags containing medium and PBL are “split” 1:2 or 1:3 to reducethe PBL concentration to a level of 1×10⁶/ml or a bit above. Forexample, a 1:2 split of cells at 2.0×10⁶/ml involves transferring 500mls of medium (containing PBL) to a new 3 liter PL732 bag and adding 500mls of AIM-V containing IL-2 to bring the total volume up to 1000 ml.The AIM-V being added to the 3 liter PL732 bag is transferred from a 10liter STAK PACK of AIM-V medium (GIBCO; Life Technologies, Grand Island,N.Y.) using a sterile Solution transfer set, Life-adapter set, 8″Interconnecting jumper tube, and a Fluid fill/weigh unit.

When PBL have been expanded beyond 3 bags (about 1500 mls each), atleast 4×10⁹ cells (generally)1×10¹° may be removed for bulk freezing ina bag, keeping at least 4×10⁹ PBL in culture in order to generate cellsfor treating the patient. For a rapidly growing culture, the PBL mightbe removed a week before treatment. However, for slower growing PBLmight not be removed for freezing until the day of harvest. PBL arecommonly used for treatment after 14-45 days in culture.

In general, PBL doubling time is 1.5 to 3 days. Thus, PBL cultures aregenerally split to new bags containing fresh medium every 3-5 days.Fungizone is left out of the last passage of cells in bags to minimizeadverse effects on the patients. Of note, a sample is collected from thelast passage of PBL for microbiology tests; this should be done 2-5 daysprior to the beginning of PBL therapy. The test takes 2 days. The bagsare then harvested for treatment using an automated process of cellharvesting as described by Muul, et al. J. Immunol. Methods 101:171,1987).

Following the last split of cells prior to use for treatment, tests aredone for bacterial and fungal contamination from samples representing10% of the bags. If treatment occurs earlier than expected, such thatFungizone is present in the PBL growth medium, PBL harvested fortreatment should be washed with 9 liters of isotonic saline, rather thanthe usual 3 liters. Cells can be infused with up to 75,000 CU IL-2 perinfusion bag.

PBL are infused in a volume of 200-300 ml of saline supplemented with 50ml of 25% human albumin (Alpha Therapeutic Co.). Cells are infused over30-60 minutes through a central venous catheter. Patients receiving dualspecificity cells are immunized with donor PBMC 1 and 8 days after eachcell infusion. Each immunization is performed with up to 5×10⁹ donorPBMC, depending on the number of cells available, administeredsubcutaneously in the thighs at a concentration of up to 7×10⁸ PBMC perml of injectate.

Cryopreserved, transduced PBL can be thawed for subsequent cycles orcourses of therapy. If necessary, repeat transduction may be performedon either fresh PBL or cryopreserved, non-transduced PBL.

IL-2 is administered at a dose of 120,000 CU/kg as an intravenous bolusover a 15 minute period every twelve hours beginning on the day of PBLadministration and continuing for up to eight doses. Doses may beskipped depending on patient tolerance. Also, if patients reach GradeIII or IV toxicity (not easily reversed) due to IL-2 except for thereversible Grade III toxicities common to IL-2 such as diarrhea, nausea,vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, orconstitutional symptoms and laboratory changes as detailed in Table 12,doses are not administered. If this toxicity is easily reversed bysupportive measures then additional doses are given. No more than 12doses of IL-2 is ever administered.

TABLE 12 Toxicity of Treatment with IL-2 Total Number of patients 652Number of courses 1039 Chills 399 Pruritus 180 Necrosis 5 Anaphylaxis 1Mucositis (requiring liquid diet) 30 Alimentation not possible 4 Nauseaand vomiting 666 Diarrhea 596 Hyperbilirubinemia (maximum/mg %) 2.1.-6.0547 6.1-10.0 179 10.1+ 83 Oliguria <80 ml/8 hours 347 <240 ml/24 hours42 Weight gain (% body weight) 0.0-5.0 377 5.1-10.0 436 10.1-15.0 17515.1-20.0 38 20.1+ 13 Elevated creatinine (maximum/mg %) 2.1-6.0 6376.1-10.0 85 10.1_(—) 10 Hematuria (gross) 2 Edema (symptomatic nerve orvessel compression) 17 Tissue ischemia 2 Resp. distress: not intubated67 intubated 41 Bronchospasm 9 Pleural effusion (requiringthoracentesis) 17 Somnolence 114 Coma 33 Disorientation 215 Hypotension(requiring pressors) 508 Angina 22 Myocardial infarction 6 Arrhythmias78 Anemia requiring transfusion (number units transfused) 1-15 377 6-1095 11-15 24 16+ 14 Thrombocytopenia (minimum/mm³) <20,000 13120,001-60,000 361 60,001-100,00 285 Central line sepsis 63 Death 10

IL-2 (Chiron), NSC #373364, is provided as a 5 mL vial containing 1.3 mgof protein as a lyophilized powder cake, with mannitol 50 mg and SodiumDodecyl Sulfate 130 μg per milligram of protein. The 1.3 mg of proteinis equivalent to approximately 21.6 million International Units (IU) or3.6 million Cetus units (CU), where 600 IU=100 CU. The vial isreconstituted with 2.0 mL of Sterile Water for Injection, USP, and theresultant concentration is 10.8 million IU/ml or 1.8 million CU/ml.Diluent should be directed against the side of the vial to avoid excessfoaming. Contents are gently swirled, not shaken, until completelydissolved. Since vials contain no preservative, reconstituted solutionshould be used with 8 hours.

Intact vials are stored in the refrigerator (2-8° C.) protected fromlight. Each vial bears an expiration date.

Reconstituted IL-2 is further diluted with 50 mL of 5% Human SerumAlbumin (HSA). The HSA is added to the diluent prior to the addition ofrecombinant IL-2. Dilutions of the reconstituted solution over a1000-fold range (i.e., 1 mg/mL to 1 μg/mL) are acceptable in eitherglass bottles or polyvinyl chloride bags. Reconstituted solutions arenot mixed with saline-containing solutions. IL-2 is chemically stablefor 48 hours at refrigerated and room temperatures, 2-30° C.

All patients receiving IL-2 also receive concomitant medications torelieve side effects as in all previous high-dose IL-2 protocols. Thefollowing concomitant medication begins the evening before the firstdose of IL-2 and continues throughout the entire cycle of treatment:acetaminophen (650 mg every 4 hr), indomethacin (50-75 mg every 6-8 hr),and ranitidine (150 mg every 12 hr). Patients receive intravenousmeperidine (25 to 50 mg) to control chills when they occur, althoughchills are unusual after the first one to two doses of IL-2.Ondansetron, droperidol, or scopolamine is available as needed for thetreatment of nausea during treatment. Steroids are not used in thesepatients and if steroids are required then the patient should be takenoff protocol therapy.

Example 12 “REP” Expansion of CTL Clones to Therapeutic Numbers

If PBL fail to expand to adequate numbers, then cultures may be expandedusing 1000 CU/ml IL-2 or the “Rapid Expansion Protocol” (REP) asdescribed below: MOv-transduced PBL are counted and the specified numberis used (Table 13). In the REP cycle immediately preceding infusion,1.25 mg/ml Fungizone and 1 ml/L Cipro are added on day 8, and AIM Vmedia is used.

On day 0 PBMC are thawed, washed twice in AIM V media, resuspended incomplete media (CM; RPMI media) and irradiated (340 Gy) as describedabove. PBMC and OKT3 are added to CM, mixed well, and aliquots aretransferred to tissue culture flasks. Viable cells are added last.Flasks are incubated upright at 37° C. in 5% CO₂.

On day 2 IL-2 is added to a final concentration of 50 CU/ml.

On day 5 20 ml (130 ml for a 175 cm² flask) of culture supernatant isremoved by aspiration (cells are retained on the bottom of the flask).Media is replaced with CM containing 50 CU/ml IL-2.

On day 8, an aliquot of cells is removed for counting and is furtheranalyzed (ELISA, FACS, etc.). If cell density is greater than 1×10⁶/ml,cells are split into additional flasks or transferred to Baxter 3 literculture bags. IL-2 is added to 50 CU/ml. Fungizone is added to a finalconcentration of 1.25 mg/ml and 1 ml/L Cipro is added.

On day 11, IL-2 is added to a final concentration of 50 CU/ml. Cells aresplit if density exceeds 1.5×10⁶ cells/ml.

On day 14, cells are harvested and either prepared for additional REPcycles or cryopreserved. In general, REP expansion of CTL clones resultsin 50-200 fold expansion. Thus, 2-3 REP cycles could be required togenerate a sufficient number of cells for patient treatment. If cellshave grown to sufficient numbers for patient treatment, a sample iscollected from each flask for microbiology tests 2-3 days before thebeginning of PBL therapy (the test takes 2 days). IL-2 is added to afinal concentration of 50 CU/ml on day 14 and every 3 days until thefinal product is prepared for infusion.

TABLE 13 Component 25 cm² flask 150 cm² flask viable transduced PBL   1× 10⁵ 1 × 10⁶ allogeneic PBMC 2.5 × 10⁷ 2 × 10⁸ OKT3 30 ng/ml 30 ng/mlCM 25 ml 75 ml AIM V 75 ml

Example 13 Assessment of Patient Response to Therapy

A complete response is defined as the disappearance of all clinicalevidence of disease that lasts at least four weeks. Partial response isdefined as a 50% or greater decrease in the sum of the products of themaximal perpendicular diameter of all measurable lesions for at leastfour weeks with no appearance of new lesions or increase in any lesions.A minor response is defined as a 25-49% decrease in the sum of theproducts of the maximal perpendicular diameters of all measurablelesions but no appearance of new lesions and no increase in any lesion.

Any patient with less than a minor response will be considered anonresponder. The appearance of a new lesion or a greater than 25%increase in the product of perpendicular diameters of any prior lesionfollowing a partial or complete response will be considered a relapse.

Example 14 Generation of Recombinant Viral Vector Encoding the MOv-γChimeric Receptor

Retroviral supernatant containing the MOv-γ chimeric receptor gene wasused for lymphocyte transductions (Retroviral supernatant to be producedby Somatix, Inc, Alameda Calif.).

For the generation of high-titer recombinant viral vector encoding theMOv-γ chimeric receptor, the MFG-S retroviral vector and the ψ-CRIPpackaging cell line were used (Danos, O. and R. C. Mulligan. Proc. Natl.Acad. Sci. U.S.A., 85:6460-6464, 1988), similar to that approved for usein ongoing clinical trials (Jaffee, et al. Cancer Res., 53:2221-2226,1993). In the MFG-S vector, Moloney murine leukemia virus (MoMLV) longterminal repeat (LTR) sequences were used to generate both a full lengthviral RNA necessary for the generation of viral particles and asubgenomic mRNA analogous to the MO-MuLV envelope RNA, which isresponsible for the expression of the MOv-γ gene (FIG. 15). The vectorretained sequences in both the viral gag region shown to improve theencapsidation of viral RNA and the normal viral 5′ and 3′ splice sitesnecessary for generation of the subgenomic RNA. Three additional pointmutations were introduced into the viral gag region to eliminate thepotential expression of two overlapping open reading frames (ORFs),which encode the NH₂ portion of both the cell surface and cytoplasmicgag-pol polyproteins. DNA sequences encoding MOv-γ were inserted suchthat the initiation codon of the inserted sequences was placed preciselyat the position normally occupied by the initiation codon for envtranslation, and minimal 3′ non-translated sequences were included inthe insert. The entire DNA sequence from LTR to LTR was determined forboth strands of the vector and no mutations or base substitutions werediscovered.

The T-CRIP cell line provided the viral proteins necessary forencapsidation of recombinant retroviral genomes into infectiousparticles. As is the case with other packaging cell lines, theexpression of the relevant viral gene products in Ψ-CRIP cells isaccomplished in a manner designed to prevent the encapsidation andmobilization of the RNA molecules encoding the viral gene products. Thismakes possible the generation of stocks of replication-deficientrecombinant virus, free of replication-competent virus.

High titer stocks of recombinant virus suitable for clinical use weregenerated from cultures derived from the working cell bank (WBC)propagated in a closed-loop perfusion system designed for the massculture of anchorage dependent cells. The system allowed the culturemedium to be monitored for perfusion rate, oxygen levels, and pH,permitting the growth and maintenance of large numbers of cells in aminimal volume of medium. In its current configuration, approximately5×10¹⁰ cells were cultured in a single vessel which minimized the riskof contamination from handling multiple flasks, and additionally ensureda consistent lot of recombinant virus.

To initiate a production run, a vial of the WCB of the producing cellline was thawed and expanded in culture to generate sufficient numbersof cells to seed the bioreactor. During this brief scale-up period, thecells were re-tested for sterility and mycoplasma contamination. Thesystem was then seeded and monitored until the optimal cell density isachieved. At this point, fresh culture supernatant was collected,filtered, and stored in frozen aliquots for quality control and safetytesting as required for FDA approval, followed by clinical use.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

1. A composition comprising a T lymphocyte having a chimeric receptor orT-cell receptor reactive with a tumor antigen and an endogenous T-cellreceptor reactive with a cell that is allogeneic to the T lymphocyte.2-3. (canceled)
 4. The composition of claim 1 wherein the tumor antigenis an ovarian tumor antigen.
 5. The composition of claim 1 wherein thetumor antigen is a melanoma antigen.
 6. (canceled)
 7. The composition ofclaim 1 wherein the chimeric receptor is a single chain Fv receptor. 8.The composition of claim 1 wherein the allogeneic cell is an allogeneicperipheral blood cell.
 9. (canceled)
 10. The composition of claim 1wherein the chimeric receptor is Mov-γ.
 11. (canceled)
 12. A lymphocytecomprising a T-cell receptor reactive with an allogeneic cell and achimeric receptor reactive with a tumor antigen, wherein the lymphocyteis activated in vivo with the allogeneic cell. 13.-14. (canceled) 15.The lymphocyte according to claim 12 wherein the allogeneic cell is aperipheral blood cell. 16.-39. (canceled)
 40. A pharmaceuticalcomposition comprising: a T lymphocyte comprising a chimeric receptorreactive with a tumor antigen and an endogenous T-cell receptor reactivewith a cell that is allogeneic to the T lymphocyte; and apharmaceutically acceptable carrier. 41.-43. (canceled)
 44. Thecomposition of claim 4, wherein the ovarian tumor antigen is folatebinding protein (FBP).
 45. The composition of claim 1, wherein the Tlymphocyte is a human T lymphocyte.
 46. The lymphocyte of claim 12,wherein the lymphocyte is a human lymphocyte.
 47. The lymphocyte ofclaim 12, wherein the tumor antigen is an ovarian tumor antigen.
 48. Thelymphocyte of claim 47, wherein the ovarian tumor antigen is FBP. 49.The lymphocyte of claim 12, wherein the chimeric receptor is Mov-γ. 50.A pharmaceutical composition comprising the lymphocyte of claim 12 and apharmaceutically acceptable carrier.
 51. A composition comprising thelymphocytes prepared by selecting for lymphocytes reactive with anallogeneic cell ex vivo; and transducing the lymphocytes with a chimericreceptor gene, said gene encoding a receptor which is reactive with atumor antigen.
 52. A composition comprising a population of Tlymphocytes comprising (a) a chimeric receptor or T cell receptor thatis reactive with a tumor antigen, and (b) a T-cell receptor that isreactive with an allogeneic cell, wherein the population of Tlymphocytes has been exposed to a cell that is allogeneic to anindividual or subpopulation of T lymphocytes of the population underconditions which expand and activate the individual or subpopulation ofT lymphocytes.
 53. The composition of claim 52, wherein the tumorantigen is an ovarian tumor antigen.
 54. The composition of claim 53,wherein the ovarian tumor antigen is folate binding protein (FBP). 55.The composition of claim 52, wherein the cell is a peripheral bloodmononuclear cell, splenocyte, a dendritic cell, or a B cell.
 56. Thecomposition of claim 52, wherein the T lymphocyte is a human Tlymphocyte.
 57. The composition of claim 52, wherein the populationfurther comprises the cell that is allogeneic to the T lymphocytes. 58.The composition of claim 51, further comprising the cell that isallogeneic to the lymphocytes.